We have utilized molecular and imaging techniques to gain new insights into the behavior of hematopoietic stem and progenitor cells (HSPCs) in vivo. Utilizing lentiviral vectors carrying genes for 5 distinct fluorescent proteins (FPs) termed LEGO vectors, we have utilized a combinatorial color approach to be able to uniquely mark and then track output from individual HSPCs in time and space in vivo. A technologically-advanced and unique imaging approach combining confocal microscopy, 2 photon microscopy and advanced analytic approaches was developed, and has been applied to study the process of hematopoietic engraftment in the marrow of mice and monkeys. Early engraftment is endosteal, and large clones consisting of the progeny of single HSPCs remain distinctly and surprisingly localized in the marrow for up to 3-4 months. This result suggests that following cell division, HSPCs spread contiguously in the marrow instead of recirculating to a new location via mobilization into the blood, and that exit of HSPCs from the marrow may be primarily a death pathway. These studies were performed utilizing total body irradiation conditioning, and new studies are ongoing asking whether the geographic patterns of engraftment and hematopoiesis may be different with alternative conditioning regimens, or no conditioning (utilizing immunodeficient/stem cell deficient recipients that can engraft all donor cells without any conditioning, now successfully re-derived from frozen embryos as required to bring new mouse strains into the NIH). Contributions of HPSC-derived differentiated progeny cells could also be examined in mice transplanted with LEGO-transduced HPSCs, at very high resolution showing clear morphology of all cell types in various tissues of interest. Intercalating HSPC-derived cells could be easily mapped in all tissues, but there was no evidence for direct contribution of HSPC-derived cells to endodermal or ectodermal tissues. In collaboration with NCI investigators we have utilized this methodology to study the in vivo distribution and behavior of mesenchymal stromal cells in the context of murine graft-versus-host disease models. We have developed lentiviral barcoding with high-diversity 31-35bp genetic barcodes to study hematopoiesis in the non-human primate model. Our collaborator Rong Lu first devised this very powerful approach and applied it to study murine hematopoiesis. We have now transplanted 8 macaques with barcoded autologous CD34+ cells, and have been able to track hematopoietic output from thousands of individual HSPCs over time (up to two years) and in multiple lineages in a quantitative and highly reproducible manner, for the first time. We have already made a number of important and novel discoveries, including the lack of evidence for a common lymphoid progenitor producing T and B cells in primates, with no shared clonal derivation of B and T cells until late after transplant, and much earlier shared clonal derivation of myeloid and B cells. We have also for the first time discovered the unique lineage derivation of the major fraction of natural killer (NK) cells. CD16+CD56- cytotoxic NK cells did not share barcodes with B, T or myeloid cells even 24 months post-transplant. In vitro and murine models have not previously been able to shed light on NK cell lineage relationships. We have continued to use barcoded cells from these macaques to further dissect in vivo NK cell ontogeny, and the process of ex vivo expansion of NK cells, highly relevant for adoptive cell therapy development, in collaboration with Dr. Rick Childs' group. We have discussed a unique self-renewing population able to regenerate CD56+ NK cells that is present in a double negative population of peripheral blood cells. We continue to search for the precursor to the ontologically-unique CD16+ mature NK subpopulation, tracking dominant clones in phenotypically purified samples from blood, bone marrow, lymph nodes, and in the future liver, vaginal and intestinal lymphoid aggregates. We have also demonstrated geographic segregation of individual HSPCs long term in specific marrow sites, confirming the findings described above using LEGO imaging techniques. The barcoding projects remain highly active, with numerous new projects shedding light on multiple aspects of hematopoiesis that can now be addressed directly in vivo via this powerful technology. We are investigating the relationship between normal HSPCs and leukemia engrafting cells using competitive repopulation in the murine model, asking whether co-infusion of increasing doses of HPSCs can compete directly with leukemic cells for marrow niches, thus slowing leukemic progression. We have data indicating competition for the same niches, with confocal imaging results also backing up these functional findings. We can also now transduce the MLL-AF9 murine leukemic cells with LEGO vectors and follow leukemic engraftment and progression in vivo in the marrow and other tissues.